Deep pain is the leading cause of long-term disability in the United States. Low back pain, fibromyalgia, and chronic regional pain syndrome are some of society?s most pervasive pain conditions, and all are characterized by deep muscle pain. Despite the high prevalence of these ailments, relatively little is known about muscle pain mechanisms. Neurons that transmit muscle pain fall under group III (lightly myelinated) and group IV (unmyelinated) classification, and innervate the connective tissue (i.e. fascia) that surrounds the muscle. Traditionally, fascia has been exclusively studied as a support structure for blood vessels and nerve fibers. Recent studies however, suggest that non-neuronal cells within connective tissue may be involved in the generation and modulation of sensory neuron activity. Fibroblasts are the main cell type in fascia. Though not electrically-excitable, these cells release various neuroactive substances and exhibit changes in membrane potential upon mechanical and chemical stimulation. Activity in neighboring sensory neurons may be initiated through these paracrine or electrical signaling mechanisms, however this has yet to be directly tested. In these experiments, optogenetics, muscle-related behavioral assays, and an ex vivo muscle-nerve recording preparation will be used to address this hypothesis. In Aim 1, the contributions of myofascial fibroblast activity on nave (i.e. un-injured) muscle sensation will be addressed. Cell-type specific optogenetic techniques will be used to modulate activity in fibroblasts; effects on muscle-related behaviors (Aim 1A) and group III/IV fiber activity (Aim 1B) will be assessed. In Aim 2, similar techniques will be employed to learn about fibroblast contributions to noxious muscle sensation. Following intramuscular Complete Freud?s Adjuvant injections, myofascial fibroblasts will be inhibited or activated in vivo to determine their role in muscle-related behaviors (Aim 2A), or in an ex vivo recording preparation to assess the ways in which they modulate group III and group IV fiber activity (Aim 2B). Collectively, these studies will provide novel insight into the contributions of connective tissue signaling in both painful and non-painful muscle sensation, potentially revealing cell populations that can be therapeutically targeted in muscle pain patients.